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In the complex interplay between neurons and glia during amyotrophic lateral sclerosis (ALS), microglia have been painted as both toxin-producing killers and protective defenders. Now a new study suggests that microglia are mere bystanders to the degeneration of motor neurons. Writing in the October 8 Journal of Neuroscience, Jean-Pierre Julien, of Laval University in Québec City, Canada, and colleagues report that when they selectively destroyed proliferating microglia in a mouse model for familial ALS, there was no effect on motor neurons or disease. However, the researchers were only able to eliminate one-third of the microglial population, leading some scientists to question the utility of the results.

Studies have pointed at glia, not the motor neurons themselves, as the culprits in familial ALS caused by mutations in superoxide dismutase (SOD1) (see ARF related news story). Microgliosis is a common feature of neurodegenerative diseases, and both astrocytes and microglia appear to be players in ALS (Yamanaka et al., 2008). Some studies suggest that in ALS microglia are the bad guys, releasing molecules, such as TNFα, that lead to motor neuron damage. Yet other studies imply microglia are good guys that produce compounds, such as IGF1, to shield neurons from further harm (Beers et al., 2008 and see ARF related news story).

Joint first authors Genevieve Gowing at Laval University and Thomas Philips of the Flanders Institute for Biotechnology in Gent, Belgium, and colleagues provide the most detailed analysis to date of what happens to microglia during disease progression in transgenic mice carrying G93A mutant human SOD1. Using flow cytometry, the authors defined three distinct cell populations expressing the microglial marker CD11b: mature microglia, myeloid precursor cells, and macrophages, which express low, medium, and high levels of CD45, respectively. The populations of these cells changed as disease progressed: mature microglia dominated in mice carrying wild-type SOD1 and in presymptomatic mSOD1 mice, outnumbering myeloid precursor cells two to one in mutant mice at 60 days. But by 120 days, there were 1.8-fold as many myeloid precursor cells as mature microglia in the transgenic animals. (Macrophages were consistently the minority.) Microglia also increased overall as symptoms developed; in the transgenic mice, spinal cord sections exhibited 1.7-fold more microglia at 120 days than at 60 days. While the data confirm that microglia proliferate in conjunction with ALS symptoms, it is not clear what the switch from mostly mature microglia to mostly myeloid precursors means, Julien said. In addition, the authors found that in end-stage mice, mature microglia carried the markers CD11c and CD86, which are characteristic of antigen-presenting cells. This suggests that the microglia may be acting to stimulate other cells of the immune system.

Gowing sought to address the role of microglia in what the authors thought would be the simplest possible way: selectively eliminate them from mSOD1 mice. If microglia are toxic, they reasoned, then eliminating them should slow the progression of the disease. Likewise, if the cells were protective, ablating microglia would only make the disease worse.

The researchers crossed the mSOD1 mice with mice that expressed herpes simplex virus thymidine kinase (TK) under the CD11b promoter. When they treated the mice with ganciclovir, TK phosphorylated it, creating a nucleoside analog. This caused cell death in CD11b+ cells that were replicating DNA. Normally, this treatment is lethal, since immune cells throughout the body express CD11b, but Gowing and colleagues got around that problem by infusing ganciclovir directly into the spinal cord. Then, using immunohistochemistry, the authors determined that lumbar spinal cord cells expressing the microglial marker Iba1 were reduced by a third, and cells expressing Mac-2, a microglial marker preferentially expressed in proliferating cells, were reduced by 50 percent.

The results were anticlimactic. Compared to single mutant mSOD1 mice, there was no significant difference in the number of motor neurons, motor axons, or innervation of the gastrocnemius in the TK crosses. Body weight and reflex scores were also similar in the TK versus control mice. “It had no effect on the disease, which is very surprising,” Julien said. He theorizes that while normal microglia could be protective, microglia carrying the mutant SOD1 are unable to defend motor neurons, and thus are rendered neutral to the disease process.

However, the large numbers of microglia still present in the TK mice weaken the study in the eyes of some scientists. “With only a 50 percent decrease in their number, he can’t conclude anything one way or the other as regards the role of microglia in the disease,” Ben Barres of Stanford University in Palo Alto, California, wrote in an e-mail to ARF. And, the remaining microglia might be sufficient to carry out whatever role they play in ALS. “Microglia are innate immune cells, and in the immune system a few autoreactive cells can control a vast cellular population,” Stanley Appel of The Methodist Hospital System in Houston wrote in an e-mail to ARF.

The authors concede that they may not have killed enough microglia, writing, “It remains possible that eliminating a larger number of SOD1-positive microglia in our model may have influenced the motor neuron degeneration.” However, Julien notes that in other studies, similar levels of microglia ablation did have significant effects, for example, exacerbating ischemic injury (see Lalancette-Hébert et al., 2007), suggesting that ALS is less sensitive to changes in microglial cell numbers. He also points out that if mSOD1 microglia were producing toxins, cutting their numbers by even a third seems likely to ameliorate disease. Based on the current and other studies, he wonders if the possibly neutral microglia are even a worthwhile drug target for ALS treatments.

Appel and others remain convinced that microglia are involved in ALS caused by mSOD1. “I think there’s no question that microglia play a role,” Appel said. Isaac Chiu of Harvard Medical School suggested that microglia might in fact be a “double-edged sword,” producing both toxic and protective factors. In designing medicines, he said, “Maybe we should focus on how to increase the protective pathways and decrease the harmful pathways.”—Amber Dance